Articles, compositions and process for cleaning surfaces by use of a catalyst at the surface

ABSTRACT

The disclosure relates to a process for the treatment of a surface with a hygiene agent which can include the steps of: a) providing at the surface a non-photochemical catalyst (such as a transition metal compound) which catalyses the formation of the hygiene agent from one or more precursors, whereby the catalyst becomes deposited at the surface, and, b) subsequently treating the surface with a treatment agent (such as a solution of hydrogen peroxide) having the or each hygiene agent precursor, such that the hygiene agent is generated at the surface. The disclosure also provides a process which includes the step of treating the surface which has a non-photochemical catalyst bound thereto with a treatment agent having at least one hygiene agent precursor which forms said hygiene agent in the presence of the catalyst and, a process for the manufacture of an article which includes the step of incorporating therein, at the time of manufacture, a non-photochemical catalyst capable of transforming at least one hygiene agent precursor into a hygiene agent.

TECHNICAL FIELD

The present invention relates to articles and a process for treatment ofsurfaces of said articles, particularly when said articles have beencontaminated with microorganisms and to compositions for treatment ofarticles. More particularly, the invention relates to a process whichfacilitates the reduction of elimination of contamination bymicroorganisms through the application of hygiene agents or precursorsthereof.

BACKGROUND OF THE INVENTION

Contamination of surfaces with microoorganisms can cause problems in anumber of areas. For example, microorganisms which are injurious tohealth may be present on a surface on which food is prepared. If theseorganisms are transferred to the food, illness can result for a personwho consumes the food. Further problems are caused by microbialcontamination of the inner walls of air-conditioning ducts or watersupply pipes within buildings, or by contamination of devices for use inor on the human body, for example, contact lenses, surgical instrumentsetc. These problems not only include the risks to health but also,especially in the case of pipes and the like, increased fluid frictionalresistance, increased heat transfer resistance and biocorrosion. Manyother examples are apparent from the literature and could be cited.

Many compositions are known and used for the cleaning and disinfectionof surfaces. Almost all these compositions share the common feature thatthey contain a hygiene agent which is toxic to or inhibits the growth ofmicroorganisms. Typical hygiene agents include, strong acids, alkali's,phenolics, oxidising agents such as peracids and hypohalides and chargedspecies such as cationic surfactants. These are generally highlyreactive species which exhibit this reactivity in terms of one or moreof, short shelf life, toxic, corrosive and irritant properties and, ingeneral, these components are required at relatively high levels informulations to obtain a satisfactory result.

In many circumstances microorganisms will grow on a surface to form aconfluent or part-confluent biofilm. Bacterial biofilms are regarded asproblematic in household and industrial hygiene as well as in medicinewhere they can act as reservoirs for spoilage organisms or pathogens orcan lead to so-called biofouling. Biofilms are regarded as problematicin infections associated with indwelling medical devices and otherarticles in contact with wet body tissues, such as contact lenses.

Conventional approaches to the control of biofilms, such as theapplication of the hygiene agents described above have demonstrated thatthese films are recalcitrant. A number of factors are thought tocontribute to this recalcitrance, and it is believed that a major factoris the ability of the glycocalyx, an extracellular polymer ofpolysaccharides and glycoproteins to react with highly reactive species,such as the above mentioned oxidising agents or halogens and to bind theabove mentioned charged species. It is believed that these interactionsprevent effective access of the hygiene agent beyond the exposed surfaceof the biofilm.

It is known that the activity of certain hygiene agents can be improvedby addition of other components. For example, the reaction ofhypochlorite solutions with strong acids will produce chlorine a potent,if somewhat dangerous, hygiene agent. It is also known to generatehygiene agents by catalytic action. EP 0436466 discloses a method ofdisinfecting a hydrogen peroxide stable material by contacting saidmaterial with a hydrogen peroxide containing solution and a hydrogenperoxide decomposition means such as a catalyst on a separate support.

WO 93/00815 discloses how it is possible to bind a photosensitiser to asurface which is capable of catalysing the formation of singlet oxygenfrom triplet oxygen under the influence of visible light, therebyproviding photobacterial properties and an autosterile character to thesurface upon exposure to visible light. Compositions for use in such amethod comprise 0.1-1.0% by weight of the photosensitiser, which may bea porphyrin or phthalocyanine, preferably in the unmetalled form. Saltsof the meso-tetra(N-octyl-4-pyridinium)porphyrin tetracation are knownto have both cytotoxic and phocytotoxic properties. Similar techniquesare known for the preparation of ceramic tiles and other such sanitaryware. In such systems, as disclosed in the trade literature of Toto Ltdof Minato-Ku, Tokyo, photoactivation of a compound based on titaniumdioxide is believed to produce an excited species which is effective asa hygiene agent.

A disadvantage of the photochemical systems is that they are lesseffective or ineffective under low light conditions or in the dark.Given that many of situations in which microbial contamination can occurare not well lit (such as the inside of air-conditioning ducts or watersupply pipes) there is a need to provide hygiene systems which areeffective against biofilms, both in lit and dark conditions withoutrequiring the use of high levels of reactive hygiene agents.

BRIEF DESCRIPTION OF THE INVENTION

We have determined that the efficacy of hygiene agents can be muchimproved if the hygiene agent is generated non-photochemically from aprecursor compound at a surface contaminated with a biofilm rather thanbeing prepared separately and applied to the exposed surface of thebiofilm. This may be accomplished by activating the surface with adark-acting catalyst prior to the growth of a biofilm and subsequenttreatment of the contaminated surface with a substrate for the catalystwhich is transformed by the catalyst into a hygiene agent.

Without wishing to limit the invention by reference to a theory ofoperation, it is believed that the production of the hygiene agent atthe surface produces a concentration gradient which diminishes away fromthe contaminated surface, rather than towards the surface as is the casewhen the agent is applied to the exposed surface of the biofilm.Moreover, consumption of the or each hygiene agent precursor at thecontaminated surface establishes a concentration gradient of saidprecursors which promotes diffusion of said precursors towards thecontaminated surface.

Accordingly, a first aspect of the present invention provides a processfor the disinfection of a surface with a hygiene agent which comprisesthe steps of:

a) providing at the surface a non-photochemical catalyst which catalysesthe formation of the hygiene agent from one or more precursors, wherebysaid catalyst become deposited at said surface, and,

b) subsequently treating the surface with a treatment agent comprisingthe or each hygiene agent precursor, such that the hygiene agent isgenerated at and disinfects said surface.

It should be noted that for certain catalysts it is not necessary thatthe above-mentioned step (a) be performed before every peformance ofstep (b) as in these embodiments there will be sufficient catalystremaining from previous cleaning cycles to produce the hygiene agentfrom the or each precursor: thus an important aspect of the presentinvention is that the process can consist of a small number oftreatments to provide the catalyst at the surface and a larger number ofsubsequent treatments to supply the catalyst with its substrate. Thusthe deposited catalyst can be used for a single disinfection operationor a plurality of such operations.

The term `non-photochemical` as used in this specification does notexclude the possibility that the properties of the catalyst will bemodified by illumination, but it is essential that the catalyst iscapable of generating the hygiene agent substantially in the absence oflight. Thus the invention has the important advantage that it enablesthe catalytic generation of hygiene agents in dark and generallyinaccessible places such as the interiors of pipes and ducts without therequirement of illumination of these places.

Accordingly, a second aspect of the present invention provides a processfor the disinfection of a surface having microbial growth thereuponwhich comprises the step of treating the surface which has anon-photochemical catalyst bound thereto with a treatment agentcomprising at least one hygiene agent precursor which forms said hygieneagent in the presence of said catalyst.

It should be noted that catalyst can be deposited at the surface byincorporation of catalytic material into the material from which thesurface is formed prior to the formation of the surface.

Accordingly, a fourth aspect of the present invention relates to aprocess for the manufacture of a three-dimensional article whichincludes the step of incorporating therein, at the time of manufacture,a non-photochemical catalyst capable of transforming at least onehygiene agent precursor into an hygiene agent effective againstmicroorganisms, wherein the article is such that in use microbial growthwill occur on a surface of the article.

A fifth aspect of the invention relates to a three-dimensional articlehaving incorporated therein, at the time of manufacture, anon-photochemical catalyst capable of transforming at least one hygieneagent precursor into an hygiene agent, wherein the article is such thatin use microbial growth will occur on a surface of the article.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a plot of hydrogen peroxide concentration vs. percentagesurviving fraction of microorganisms (both total and attached).

FIG. 2 is a plot of potassium monopersulfate (KMPS) concentration vs.percentage surviving fraction of microorganisms (both total andattached).

FIG. 3 is a graph demonstrating the effect of hydrogen peroxide on thereduction exponent k.

FIG. 4 is a graph demonstrating the effect of KMPS on the reductionexponent k.

DETAILED DESCRIPTION OF THE INVENTION

It is an essential feature of the present invention that the hygieneagent is formed by treatment of the catalyst with the or each precursorfor a hygiene agent. In certain embodiments of the present invention,these precursors themselves have anti-bacterial or other anti-microbialproperties. These properties may be exhibited to a lesser or greaterextent than those of the hygiene agent. In other embodiments theprecursors have is substantially no antimicrobial properties and, it ispreferable that, to the advantage of the user, they exhibit none orsignificantly less of the toxic, corrosive and/or irritant propertiesmentioned above.

Hygiene Agents and Catalysts

As mentioned above the treatment agent contains at least one hygieneagent precursor which forms said hygiene agent in the presence of saidcatalyst. In the context of the present invention the term hygiene agentincludes both biocidal and non-biocidal species. While it is preferablethat the hygiene agents are biocidal it is also envisaged that thehygiene agents can function in a more general sense by physical removalof at least a part of the microbes from the surface for example byweakening the attachment of the microbes to the surface or by thegeneration of gas bubbles at the surface. Thus in certain embodiments ofthe invention the hygiene agent is effective against micro-organisms byvirtue of physical rather than chemical effects.

Suitable hygiene agent precursors include peroxy compounds,isothiazolones, halides and hypohalites. It is preferable that theseprecursors are presented as a liquid composition and it is particularpreferred that said liquid composition should be essentially free of thecatalyst.

Suitable peroxy compounds include, hydrogen peroxide, sodium peroxide,peracetic acid, performic acid and mono-persulphate salts. In additionto the above-mentioned acids the use of imidoperoxy--carboxylic acidsincluding e-N-N-phthaloyl-amino-peroxy-caproic acid is envisaged. Otherperoxy-carboxylic acids, are known from a publication entitled "TAED andnew peroxycarboxylic acids as highly efficient bleach Systems", 80thAOCS Meeting, Cincinnati Ohio, May 1989 and are incorporated herein byreference. Hydrogen peroxide and potassium monpersulphate areparticularly preferred as the peroxy compounds.

Suitable catalysts for the decomposition of peroxy compounds includetransition metals and compounds thereof. It is preferred to usecompounds of the transition metals rather than the metals per se.Preferred transition metals are cobalt, copper, platinum, titanium,manganese, vanadium, silver, zinc, palladium, iridium and iron.Particularly preferred catalysts include cobalt, manganese and coppercompounds, more particularly manganese dioxide and copper or cobaltphthalocyanine. These and other catalysts can be applied by treating thesurface with a composition containing the catalyst so as to deposit saidcatalyst on the surface.

We have found that the combination of cobalt phthalocyanine as thecatalyst and potassium monopersulphate as the hygiene agent isparticularly effective in reducing the viable cell count of biofilmpopulations when said biofilms are grown on plastics materialscomprising cobalt phthalocyanine and subsequently treated with asolution containing potassium mono-persulphate.

The preparation of stable solutions of peroxy-compounds and hypohalitesis well documented in the art. It is known that these solutions shouldpreferably be free of the abovementioned metals as these are known tosignificantly reduce the shelf life of the solutions. We have found thatthe provision of the catalyst at the surface to be disinfected enableseffective results to be obtained with very low concentrations of thetreatment agent and significantly lower levels than would be requiredwithout the use of the catalyst. Thus, for example, in the case wherethe treatment agent comprises hydrogen peroxide and the catalystcomprises a transition metal, we have established that substantialremoval of biofilm may be achieved using hydrogen peroxideconcentrations of less than 3 mg/ml. Such concentrations were totallyineffective for removing and disinfecting similar biofilms from surfacesnot provided with a catalyst. It is therefore a significant advantage ofthe invention that lower concentrations of the precursor may be used,particularly, for example, in surgical or medical applications.

The invention is not limited to the use of such low levels of hygieneagent precursor in the treatment agent since, in some circumstances, theuse of higher levels may be necessary or desirable. Generally, theconcentration of the hygiene agent precursor will be from 10 ppm to 40%w/v.

Alternative catalysts include enzymes. Particularly preferred areenzymes of the `oxidase` class which produce hydrogen peroxide whenexposed to a suitable substrate.

One class of enzymes envisaged for use in the present invention are theglucose oxidases, although it is envisaged that other enzymes such asthe haloperoxidases could also be used.

The process of the invention is particularly useful when applied toceramic, glass and other such hard surfaces and, in particular, toplastics and other polymeric materials (including kitchen laminates)which, unlike certain metals, do not have the inherent ability tocatalyse the conversion of precursors to hygiene agents. While theinvention is generally described herein with reference to generalhousehold and industrial surface cleaning the application of the processof the invention to specialist cleaning tasks such as the cleaning andsterilisation of medical appliances including contact lenses anddentures, or medical or personal care apparatus including toothbrushesis not intended to be excluded.

As noted above, in certain embodiments of the invention it is envisagedthat the catalyst is incorporated into articles or applied to thesurface thereof in the process of manufacture of the articles. It ispreferable that the surface as manufactured incorporates the catalyst.Accordingly the present invention extends to many articles of laboratoryand household equipment including pipes and tubes, racks and containers,tools and utensils, including wipes.

Preferred articles are manufactured of a plastics material which has thecatalyst distiributed therein. Alternatively, the catalyst can beapplied to the surface of the article in the form of a coating.

It is particularly preferred that the treatment agent is a liquid whichcomprises an aqueous carrier and one or more precursors of the or eachhygiene agent in solution. It is preferred that the one or moreprecursors of the or each hygiene agent forms a stable solution orsuspension at a level of up to 1% in water, i.e. gaseous precursors aregenerally avoided and solid or liquid precursors are preferred. Theliquid precursors can be in the form of mists, sprays and aerosols. Onegaseous hygiene agent which is envisaged as useful in the practice ofthe present invention is hydrogen peroxidse in a vapourised form. Thisvapour is believed to be highly reactive and to require specialprecautions to be taken when it is used at conventional concentrations.It is believed that the present invention provides for the use ofeffective levels of hydrogen peroxide vapour which are lower than theconventional concentrations. Similarly, it is believed that organicacids in vapour form can be employed.

Surfactants

It is preferred that the treatment agent further comprises a surfactant.Surfactants can be nonionic, anionic, cationic, amphoteric orzwitterionic provided that they, and where appropriate theircounter-ions, do not react substantially with the hygiene agent or itsprecursor.

Suitable nonionic detergent active compounds can be broadly described ascompounds produced by the condensation of alkylene oxide groups, whichare hydrophilic in nature, with an organic hydrophobic compound whichmay be aliphatic or alkyl aromatic in nature. The length of thehydrophilic or polyoxyalkylene radical which is condensed with anyparticular hydrophobic group can be readily adjusted to yield awater-soluble compound having the desired degree of balance betweenhydrophilic and hydrophobic elements.

Particular examples include the condensation product of aliphaticalcohols having from 8 to 22 carbon atoms in either straight or branchedchain configuration with ethylene oxide, such as a coconut oil ethyleneoxide condensate having from 3 to 10 moles of ethylene oxide per mole ofcoconut alcohol; condensates of alkylphenols whose alkyl group containsfrom 6 to 12 carbon atoms with 3 to 10 moles of ethylene oxide per moleof alkylphenol.

The preferred alkoxylated alcohol nonionic surfactants are ethoxylatedalcohols having a chain length of C9-C11 and an EO value of at least 3but less than 10. Particularly preferred nonionic surfactants includethe condensation products of C₁₀ alcohols with 3-8 moles of ethyleneoxide. The preferred ethoxylated alcohols have a calculated HLB of10-16. An example of a suitable surfactant is `IMBENTIN 91-35 OFA` (TM,ex. Kolb AG) a C₉₋₁₁ alcohol with five moles of ethoxylation.

When present, the amount of nonionic detergent active to be employed inthe composition of the invention will generally be from 0.1 to 30% wt,preferably from 1 to 20% wt, and most preferably from 3 to 10% wt fornon-concentrated products. Concentrated products will have 10-20% wtnonionic surfactant present, whereas dilute products suitable forspraying will have 0.1-5% wt nonionic surfactant present.

Minors and Optional Components

The composition according to the invention can contain other minor,inessential ingredients which aid in their cleaning performance andmaintain the physical and chemical stability of the product. Typically,these will be materials known in the art of formulation of cleaningcompositions.

For example, the composition can contain detergent builders. In general,the builder, when employed, preferably will form from 0.1 to 25% byweight of the composition.

Optionally, the composition can include one or more amphotericsurfactants, preferably betaines, or other surfactants such asamine-oxide and alkyl-amino-glycinates. Betaines are preferred forreasons of cost, low toxicity (especially as compared to amine-oxide)and wide availability. These components should be selected such thatthey do not react with the hygiene agent precusor or other components ofthe product.

Metal ion sequestrants, including ethylenediamine-tetraacetates,aminopolyphosphonates (such as those in the DEQUEST^(R) range) andphosphates and a wide variety of other poly-functional organic acids andsalts, can also optionally be employed. Again, these components shouldbe selected such that they do not react with the hygiene agent precusoror other components of the product.

Hydrotropes, are useful optional components. Suitable hydrotropesinclude, alkali metal toluene sulphonates, urea, alkali metal xylene andcumene sulphonates, polyglycols, >20EO ethoxylated alcohols, shortchain, preferably C₂ -C₅ alcohols and glycols. Preferred amongst thesehydrotropes are the sulphonates, particularly the cumene, xylene andtoluene sulphonates. Again, these components should be selected suchthat they do not react with the hygiene agent precusor or othercomponents of the product.

Typical levels of hydrotrope range from 0-5% for the sulphonates.Correspondingly higher levels of urea and alcohols are required.Hydrotropes are not always required for dilute, sprayable products, butmay be required if lower EO or longer alkyl ethoxylates are used or thecloud point needs to be raised considerably. The cumene sulphonate isthe most preferred hydrotrope.

Compositions according to the invention can also contain, in addition tothe ingredients already mentioned, various other optional ingredientssuch as, solvents, colourants, optical brighteners, soil suspendingagents, detersive enzymes, compatible bleaching agents, gel-controlagents, freeze-thaw stabilisers, further bactericides and orantimicrobials, perfumes and opacifiers. It is also envisaged that thecompositions according to the invention can be delivered in anencapsulated form such that there is a time-delayed release of eitherthe catalyst or the treatment agent at the surface.

A number of non-limiting applications in which it is envisaged that theinvention can be used are given below:

a) Industrial, institutional and domestic cleaning and/or disinfectionof hard surfaces to which the catalyst is applied including metal,plastics materials or other polymers, ceramic, and glass surfaces usedfor the preparation of food and beverages (e.g. worktops, conveyorsystems and utensils) or other industrial, institutional and domesticsurfaces such as sanitary ware.

b) Industrial, institutional and domestic fluid supply applications,e.g. provision of the catalyst on the inner wall of a pipe (e.g asplastics pipe) used for supplying drinking water, or the inner wall ofan air conditioning duct, or the inner wall of an oil pipeline. Furtherapplications of this category include provision of the catalyst in heatexchangers and radiators so as to permit disinfection thereof andprevent biofouling. In these instances disinfection is effected byflowing the treatment agent through the installation concerned.

c) Applications in disinfection of medical, surgical or dentalapparatus, equipment, facilities or supplies e.g. provision of thecatalyst on a surface of a catheter, contact lens, surgical dressing orsurgical instrument (e.g. an endoscope) so as to permit readydisinfection thereof.

d) Applications in coating technology, e.g. by incorporation of thecatalyst in a paint varnish or other coating composition to be appliedto a surface so that the surface can be subsequently disinfected.

e) Horticultural applications, e.g. for sterlising the surfaces ofgreenhouses using low concentrations of the precursor material.

f) Marine applications to treat biofouling of submersed portions ofvessels or structures.

g) Soft surfaces including fabrics (eg. in dressings, wipes and cloths),and non-living materials of biological origin (such as wood).

h) Applications relating to personal washing of skin and hair andpersonal oral care processes and articles, including tooth-brushes.

In order that the invention may be further understood it will bedescribed hereinafter with reference to the following illustrativeexamples:

EXAMPLES Example 1 Use of manganese dioxide catalyst in surfacetreatment

Sufficient polystyrene foam was dissolved in a small quantity ofchloroform to provide a thin solvent-based cement. Into this cement wasmixed a minor amount of manganese dioxide. The resulting product wasapplied to glass cover-slips and allowed to dry. In a similar manner,glass cover slips were partially coated with an identical cementcomposition which did not contain the manganese compound.

After the chloroform had evaporated, both sets of slips were immersed ina Luria broth culture of E. coli (approximately 10⁷ cells/ml. The brothwas maintained in a heated waterbath so as to allow a biofilm to becomeestablished on the surface of the coverslips. Typical biofilms producedin this manner have a cell density of around 10⁶ cells/cm².

After the biofilm had been produced, the coverslips were removed fromthe broth and treated with solutions of hydrogen peroxide. It was noted,by inspection, that this process removed significantly more of thebiofilm from the slip to which the manganese dioxide containing cementhad been applied, as compared with the slips which had simply beencoated with the cement. It was also noted that the production of oxygenby the decomposition of hydrogen assisted in the removal of the biofilmfrom the surface.

Example 2 Use of copper and cobalt phthalocyanin catalysts inmanufacture.

Trylon (TM) Resin (polyester resin in styrene monomer, ex. Trylon Ltd,Wollaston, Northants, UK) was mixed with Trylon (TM) Hardener (methylethyl ketone peroxide) at a hardner concentration of about 1%, poured in50 ml boiling tubes and allowed to harden overnight at room temperature.Rods of plastics material were extracted by breaking the tubes.

Copper phthalocyanine (ex Sigma.) was incorporated into certain batchesof the plastics material by adding ground catalyst to 1 ml of the resin,mixing and adding the balance of the components to obtain a plasticsmaterial comprising 100 micrograms/liter of the catalyst. Similarexperiments were performed with cobalt phthalocyanine.

Disks of plastics material 1 mm thick and 20 mm in diameter were cutfrom the rods. Prior to use all disks were either placed in media andautoclaved at 121 Celcius for 20 minutes to sterilise both disks andmedia, or sterilised by storage in ethanol (70% wt vol).

The effect of the presence of the catalyst was evaluated by the effecton biofilms obtained from a stable, mucoid, clinical isolate ofPseudomonas aerucinosa PaWH. Cultures were maintained on Tryptone AoyaAgar (TSA, Oxoid (TM) CM131) slants, in the dark, at 4 Celcius, afterovernight incupation at 37 Celcius.

Where required, overnight cultures were prepared from the slopes byinoculating 100 ml volumes of Tryptone Soya Broth (TSB, Oxoid CM129),contained in 250 ml Erlenmeyer flasks, and incubating at 37 Celcius for16 h in an orbital incubator (200 rpm). 20 ml volumes of sterile salinecontaining a range of H₂ O₂ concentrations (0 to 3.75 mg.ml-1) wereinoculated with an overnight culture of Ps. aeruginosa in such a mannerthat a final concentration of 10⁸ colony forming units (c.f.u) per mlwas obtained. Test suspensions were left at room temperature for 30minutes. After exposure, the suspensions were serially diluted toneutralise the biocide and 5 replicates were plated out for eachdilution, to determine numbers of surviving cells.

Trylon discs with or without catalyst incorporated within them wereclamped vertically within a cassette made out of Teflon. The cassetteholds up to 16 discs in a radial arrangement. When immersed in culturemedium (200 ml) and contained within a 500 ml beaker, the cassetteallowed the free circulation of liquid to the discs. Beakers (500 ml)containing TSB (200 ml) and empty cassettes were sterilised byautoclaving and sterile discs were then added aseptically. Attachment ofmicroorganisms was initiated by inoculation of the growth medium with acolony taken from a Tryptone Soya Agar plate. The device was incubatedin a shaking incubator (37° C., 200 rpm) for 24 hours.

Discs, with their associated biofilms (above) were removed from thecassette, rinsed in two successive volumes (20 ml) of sterile saline (toremove loosely associated cells) and immersed in biocide (20 ml) for 30minutes at room temperature. After exposure the discs were rinsed in twosuccessive volumes (20 ml) of sterile saline (rinse n°1), andtransferred to a sample tube containing 10 ml sterile saline. Sodiumthiosulphate at 3.75 mg/ml was employed instead of sterile saline whenthe hygiene agent precursor was KMPS.

The tubes were then shaken vigorously for 10 minutes in a flask shaker(Griffin and George Scientific, London, UK) in order to remove themajority of the biofilm cells. It is thought that this process removesthose cells that are attached only to other cells, while leaving thosethat are firmly attached to the test surface. The rinses and vigorousshaking in diluent of the biofilms serves to further neutralise (whererequired) and stop the action of the biocide. Discs were removed andfurther rinsed in three successive 20 ml volumes of sterile saline(rinse n°2). Finally, the discs were transferred to a succession of 15TSA plates. These plates were incubated and viable counts made. Viablecounts were also performed on both rinse solutions, on the shakesolution, and the residual solution from the biocide treatment vial.These viable counts were combined to give an estimate of the survivingpopulation that could be rinsed-off and/or removed from the biofilm byshaking after treatment. The results from the plate succession indicatenot only the numbers of viable cells remaining firmly in associationwith the test surface, but also the ease with which they can be removedby friction.

Colonies associated with the plate succesisions arose from cells whichhad resisted removal either by rinsing or vigorous shaking in saline.Accordingly, results were divided into two categories: (1) effect ofbiocide on attached biofilm populations, derived from the numbers ofc.f.u. transferred, from three replicate discs, to each succession of 15TSA plates, and (2) effect of biocide on total biofilm populations,expressed as the sum of attached populations and of those organismsremoved during rinsing and shaking after exposure to biocide.

After estimation of the number of surviving cells by the platesuccession method the mean c.f.u. decreased exponentially with platesuccession number. Regression analysis was performed using an SPSSstatistics package, and lines of best fit determined. Data was fitted toequation 1:

    CFU=A.10.sup.-KN {1}

where:

CFU is the number of organisms transferred to any given TSA plate,

A is a constant corresponding to the intercept value of the best fitline with the ordinates axis,

K is the reduction exponent of the best fit line, and is believed togive a measure of the strength of attachment to the surface,

N is the plate succession number

From equation 1, and with known A and K values, it was possible tocalculate the total number of organisms on each disc.

Similar experiment were performed with potassium monopersulphate as analternative to the hydrogen peroxide biocide.

Table 1 below gives the concentration of biocide in micrograms/mlrequired to reduce by 90% the numbers of viable cells recovered fromattached biofilms on Trylon disks containing either copper or cobaltphthalocyanine as determined by the above described agar successionmethod, when exposed to hydrogen peroxide and potassium monopersulfate(KMPS).

                  TABLE 1                                                         ______________________________________                                                       H.sub.2 O.sub.2                                                                    KMPS                                                      ______________________________________                                        Cu-catalyst             2200                                                                                 374                                              Co-catalyst                 150                 5                           ______________________________________                                    

Table 1 shows that KMPS was generally more effective against attachedbiofilm populations on catalyst containing disks than was hydrogenperoxide and that the KMPS/cobalt phthalocyanine combination was themost effective of the combinations tested.

Table 2 below gives the concentration of biocide in micrograms/mlrequired to reduce by 90% the numbers of viable cells recovered fromtotal biofilm populations and, where specified, plantonic populations onTrylon disks containing either copper or cobalt phthalocyanine, whenexposed to hydrogen peroxide and potassium permonosulphate.

                  TABLE 2                                                         ______________________________________                                                        H.sub.2 O.sub.2                                                                   KMPS                                                      ______________________________________                                        Cu-catalyst       375   200                                                     Co-catalyst               125                  3.75                           Planktonic               1125               1200                            ______________________________________                                    

From the results in Table 2 it can be seen that KMPS appears to besignificantly more affective against the total biofilm population thanhydrogen peroxide. The KMPS/cobalt phthalocyanine combination achieved90% reduction in viable cells at concentrations of up to 100 times lowerthan for other combinations.

We have determined that, at a concentration of 3 mg.ml-1, there is a 1.5log cycle decrease in the percentage survival of biofilm cells attachedto cobalt phthalocyanine containing discs whereas those cells grown andtreated with hydrogen peroxide on Trylon alone displayed no measurabledecrease in viability. It was also noted that hydrogen peroxidetreatment increased the value of the blot succession reduction exponentdemonstrating that, not only was there an increase in hydrogenperoxide-mediated killing, but also that those cells surviving exposureto hydrogen peroxide were more loosely attached to the surface and as aresult more easily removed.

The plate succession method enables the effects of the biocide on thecomplete original biofilm population to be estimated, by addition of theCFU obtained to the estimated residual attached population. Viability oftotal biofilms decreased to a much greater extent (3 log cycles) thanthat of the residual attached populations and, at a concentration of 3mg.ml-1 hydrogen peroxide, to the same extent as that of planktonicpopulations. This can be explained by the fact that cells removed duringrinse and shake steps are those cells that are more loosely attached tothe substratum, or bound only to other cells. These cells do notnecessarily benefit from the protection afforded by biofilm mode ofgrowth per se (altered physiology, encasement within a matrix ofextracellular polymeric substance (EPS) hindering diffusion of biocide)and thus would be more susceptible to the biocidal effects of hydrogenperoxide.

The results of the treatments with the biocide solutions are shown inFIGS. 1-4 of the accompanying drawings. In the graphs presented, the CFUdata were normalised with respect to the discs which were exposed tozero concentrations of the biocide.

FIG. 1 is a plot of hydrogen peroxide concentration vs. percentagesurviving fraction of microorganisms (both total and attached). Thecontrol biofilms established on the Trylon discs i.e. without catalystwere unaffected by the concentration of hydrogen peroxide employed. Incontrast both the total and attached populations were substantiallyreduced on the copper and cobalt containing discs. As can be seen fromthe graphs cobalt was significantly better in its enhancement ofbacterial kill than copper.

FIG. 2 is a plot of KMPS concentration vs. percentage surviving fractionof microorganisms (both total and attached). The control biofilmsestablished on the Trylon discs i.e. without catalyst were unaffected bythe concentration of KMPS employed. In contrast both the total andattached populations were substantially reduced on the copper and cobaltcontaining discs. As can be seen from the graphs cobalt was particularlyeffective in enhancing bacterial kill at very low concentrations ofKMPS.

FIG. 3 is graph demonstrating the effect of hydrogen peroxide on thereduction exponent k. FIG. 4 is similar but shows the results for KMPS.These graphs clearly demonstrate that the microorganisms which survivedthe disinfectant tratment were less firmly attached after treatment on acatalyst containing material and would therefore be more easily removedby normal cleaning procedures. The attached survivors became morereadily removed with increasing biocide concentrations.

We claim:
 1. A process for the disinfection of a surface with a hygieneagent which comprises the steps of:a) providing at the surface anon-photochemical catalyst for catalyzing the formation of an hygieneagent from one or more precursors, whereby said catalyst becomesdeposited at said surface, and, b) subsequently treating the surfacewith a treatment agent comprising at least one hygiene agent precursor,such that the hygiene agent is generated at and disinfects said surface.2. A process for the disinfection of a surface having microbial growththereupon which comprises the step of treating the surface which has anon-photochemical catalyst bound thereto with a treatment agentcomprising at least one hygiene agent precursor which forms saidtreatment agent in the presence of said catalyst.
 3. A process for themanufacture of a three-dimensional article which includes the step ofincorporating therein, at the time of manufacture, a non-photochemicalcatalyst capable of transforming at least one hygiene agent precursorinto an hygiene agent, wherein the article is such that in use microbialgrowth will occur on a surface of the article.
 4. A process according toclaim 1 wherein the hygiene agent and/or the at least one hygiene agentprecursor is a peroxy compound, isothiazolone, halide or hypohalite. 5.A process according to claim 1 wherein the hygiene agent and/or the atleast one hygiene agent precursor is hydrogen peroxide, sodium peroxide,peracetic acid, performic acid, a monopersulphate salt or animidoperoxy-carboxylic acid.
 6. A process according to claim 1 whereinthe non-photochemical catalyst is a transition metal or compoundthereof.
 7. A process according to claim 1 wherein the concentration ofthe at least one hygiene agent precursor in the treatment agent is atleast about 10 ppm.
 8. A three-dimensional article having incorporatedtherein, at the time of manufacture, a non-photochemical catalystcapable of transforming at least one hygiene agent precursor into anhygiene agent effective against microorganisms, wherein the article issuch that in use microbial growth will occur on a surface of thearticle.
 9. A process according to claim 1 wherein the concentration ofthe at least one hygiene agent precursor in the treatment agent is lessthan about 40% w/v.
 10. A method of removing microorganisms from asurface comprising:a) providing a surface having a non-photochemicalcatalyst associated therewith; b) placing the surface in an environmentthat leads to microorganism growth on at least a portion of the surface;and c) contacting at least a portion of the surface with a hygiene agentprecursor such that a hygiene agent is generated by catalysis at thesurface.
 11. The method according to claim 10 wherein the step ofcontacting the surface with the hygiene agent precursor occurs when thesurface is in the same location as when the surface was in theenvironment leading to microorganism growth.